Selection and use of cold-tolerant bacillus strains as biological phytostimulators

ABSTRACT

The invention relates to a biological product for increasing the yield of crop plants. The invention can be used in agriculture, horticulture and plant protection. The product for stimulating the growth of crop plants is characterized by containing a cold-tolerant  Bacillus  strain.

The invention relates to a biological means for improving the yield ofcultivated plants. The areas of application of the invention areagriculture, horticulture and plant protection.

STATE OF THE ART

The introduction of biological plant protection and organic fertilisersis an environmentally, friendly alternative to chemical pesticides andmineral fertilisers. “Plant-growth-promoting” rhizobacteria (PGPR) areused for the production of effective organic formulae. Members of theBacillus type with the ability to form permanent endospores are of greatimportance since they have a natural high degree of stability. Sporesare permanent forms of the bacterial cell, which can withstand extremeenvironmental influences such as heat and aridity, and can be stored foryears) without a loss of activity.

Organic formulae of members of the Bacillus type are the most frequentlyused biological plant protection and phytostimulators (Borriss 2011).Aside from the known producers of the insecticides Bacillusthuringiensis and Bacillus kurstakii, these are strains of the Bacillussubtilis, family incl. B. amyloliquefaciens subsp. plantarum, B.subtilis, B. licheniformis and B. pumilus, which are most commonly usedin this field. Examples of commercial products (organic fertilizersand/or plant protection) that contain B. amyloliquefaciens plantarumspores are Kodiak™ (Bayer Crop Science), Companion (Growth ProductsLtd.), BioYield™ (Bayer Crop Science), INTEGRAL® (BASF), VAULT® (BASF),SERENADE Max® (Bayer Crop Science), CEASE® (BioWorks, Inc.), RhizoVital®(ABiTEP GmbH), FZB24® (ABiTEP GmbH), Double Nickel 55™ (Certis U.S.A.),Amylo-X® (Certis U.S.A.). Plant protection based on B. licheniformis areGreen Releaf and EcoGuard (Novozyme Biologicals Inc.). The B. pumilusGB34 strain (Yield Shield, Bayer Crop Science) is an active ingredientof organic fungicides. Other EPO-registered B. pumilus organicfungicides are SONATA (Bayer Crop Science), and GHA 180 (PremierHorticulture). Members of B. mojavensis have also been described asphotostimulatory and capable of endophytic growth (Bacon & Hinton 2002).Other bacilli which do not belong to the B. subtilis species complexalso stimulate plant growth and plant health. B. firmus GB126 (BioNemAgroGreen, Israel now taken over by Bayer Crop Science) is used as anEPO-registered nematicide for the organic control of phytopathogenicnematodes. A similar product from B. firmus Bmj WG is currently beingdeveloped by Certis U.S.A. A phytostimulatory B. megaterium (López-Bucioet al. 2007) strain is the basis for the BioArc organic fungicide.Members of the Bacillus cereus species complex, e.g. B. cereus UW8(Handelsman et al. 1990), B. thuringiensis and B. weihenstephanensishave also been described as plant growth promoting, endospore-formingrhizobacteria.

These bacteria used for producing the current organic products are knownas “mesophile” bacteria and require growth temperatures of over 15° C.in order to develop their effect as phytostimulators. More than 85% ofthe Earth's surface is covered by cold ecosystems, however, includingAlpine mountain regions and areas that are oceanic and close to thepoles (Feller & Gerday 2003). At temperature conditions below 15° C.,which are typical for these regions, mesophile bacteria can have nopositive effect on plant growth. An attractive alternative is the use ofcold-tolerant microorganisms. Although their optimal growth conditionslie in the mesophile range, these microorganisms have developedmechanisms which give them the ability to continue to grow at lowertemperatures (≤10°-0° C.) (Kumar et al. 2010). A note was made that theuse of cold-tolerant and plant growth promoting bacteria as inoculants,in particular for agricultural production in tempered climate zones withlow temperatures and a short growth season can be attractive (Mishra etal. 2012). However, the known examples for the use of cold-tolerantbacteria in plant production on gram-negative phytostimulatory bacteriasuch as Pseudomonas (Katiyar & Goel 2003), Burkholderia (Barka et al.2006), Acinetobacter (Gulati et al. 2009), Azospirillum (Kaushik et al.2002), Rhizobium (Prevost et al. 2003), Bradyrhizobium (Zhang et al.2003), Pantoea (Selvakumar et al. 2008a), Serratia (Selvakumar et al.2008b). Although in the past, several psychrophile or cold-tolerantBacillus types, such as Bacillus globisporus (now Sporosarcinaglobisporus), Bacillus insolitus (now: Psychrobacillus insolitus),Paenibacillus macquariensis, Bacillus marinus (now: Marinibacillusmarinus), Bacillus psychrophilus (now: Sporosarcina psychrophilus),Bacillus psychrosaccharolyticus, Bacillus psychrotolerans (now:Psychrobacillus psychrotolerans) and Bacillus psychrodurans (now:Psychrobacillus psychrodurans) have been described (El-Rahman et al.2002, Krishnamurti et al. 2010), there is a lack of informationregarding Bacillus strains with a phytostimulatory effect.

GOAL AND OBJECTIVE OF THE INVENTION

The goal of the invention is to develop an environmentally sensitive andsustainable means for stimulating plant growth, including attemperatures below 15° C. The means should in particular be used inclimatically unfavourable regions such as mountain regions and areasrelatively close to the poles of the northern and southern hemisphere.

NATURE OF THE INVENTION

The object is attained according to the claim; subclaims 2-11 arepreferred variants. The core of the invention is the use ofcold-tolerant bacillus strains as biological phytostimulators. The scopeof the invention further includes the combination of cold-tolerantbacillus strains with mesophile bacteria of the order Bacillales,preferably of type Bacillus and Paenibacillus, and the combination withhumic acids. In the search for plant growth-promoting bacteria that showclear growth at temperatures below 15° C., it was found thatendospore-forming bacilli that were isolated from cool climate regionssuch as mountain regions in the uplands of Tibet (People's Republic ofChina) or the Alpine foothills (1400 m) are also able to grow attemperatures below 12° C. In accordance with their 16S rNA sequences,these cold-adapted bacteria were assigned to the types Bacillus simplex,Bacillus pumilus and to the Bacillus subtilis species, complex. Due totheir gyrA and cheA sequences, the members of the B. subtilis speciescomplex were identified as belonging to the type Bacillus atrophaeus.The plant growth-promoting effect was examined with Arabidopsisseedlings in potted experiments. Cold-tolerant members of the typesBacillus simplex, Bacillus atrophaeus and Bacillus pumilus showed aclear phytostimulatory effect (example 6). The B. simplex (ABI02S1), B.atrophaeus (ABI02A1), and B. pumilus (ABI02P1) strains were selected asexamples for further examinations. These strains are described below:

Bacillus atrophaeus differs from B. subtilis through its DNA sequenceand the formation of a dark pigment (Nakamura 1989). Its ability topromote plant growth has been documented (Karlidag et al. 2010, Ertuk etal. 2011, Chan et al. 2013). The taxonomic affiliation of the strainABI021A1 with Bacillus atrophaeus was determined by comparing its 16SrRNA, qyrA and cheA partial sequence (see example 1):

TABLE 1 Gene Primer fragment Species Accession BLAST pRB1601 16SrRNA B.atrophaeus 1942 CP002207.1 451/453 (99%) pRB1602 16SrRNA B. atrophaeus1942 CP002207.1 470/473 (99%) gyrFW gyrA B. atrophaeus 1942 CP002207.1863/872 (99%) gyrRV gyrA B. atrophaeus 1942 CP002207.1 857/865 (99%)cheAFW cheA B. atrophaeus 1942 CP002207.1 604/698 (87%) cheARV cheA B.atrophaeus 1942 CP002207.1 509/533 (95%)

ABI02A1, ABI03 and ABI05 are characterised by the following properties:

The physiological properties of BI02A1, ABI03 and ABI05 are shown inapplication example 3. The strains promote plant growth (see applicationexample 1) and suppress the growth of bacterial (Xanthomonas oryzae) andfungal (Sclerotinia sclerotorum) pathogens in vitro. ABI02A1 was storedas strain DSM 32019, ABI03 was stored as strain DSM 32285 and ABI05 wasdeposited as strain DSM 29418 at the DSMZ, Braunschweig. Bacillusatrophaeus has already been described as a plant growth-promotingbacterium (Bai et al. 2002). To date, no reports have been made of aphytostimulatory effect at low temperatures with regard to B. simplex,however.

Bacillus simplex ABI02S1 and ABI12 already grow at 4° C. The growthmaximum is 45° C. The physiological properties of the strain are shownin illustrative representation 4. Members of the Bacillus simplexspecies have currently been identified as phytostimulatory bacteria(Schwartz et al. 2013). To date, no reports have been made of a growthstimulation effect at low temperatures. The ABI02S1 strain was depositedas DSM 32020 and the ABI12 was deposited as DSM 32283 at the DSMZBraunschweig, Germany.

The invention will now be explained in greater detail with reference toexamples.

ILLUSTRATIVE EMBODIMENTS Example 1: Isolation of Potential Cold-TolerantBacteria

Cold-tolerant bacillus strains were isolated e.g. from the uplands ofthe Autonomous Province of Tibet and the Alpine foothills, height 1,400m. The Tibetan mountainous region lies at an average altitude of 4,000 mand has an annual average temperature of 10° C. Typical for this regionare the large differences in temperature between day and night. Thesamples were either removed directly from plant roots or from theadhered earth:

2.5 g sample material were re-suspended in 25 g of distilled water for 2h under continuous shaking. To kill vegetative cells, the suspension wasthen incubated for an hour at 80° C. 10 ml of the suspension were addedto 40 ml of a mineral-salt medium and incubated for one week at 20° C.until under microscopic monitoring, rod-shaped cells emerged. Then, thesuspension was diluted to 10⁻¹ to 10⁻⁵ and plated on minimal agar. Theplates were incubated at 20° C. and the colonies formed were separatedon nutrient agar or LB agar.

The taxonomic classification of the isolates was achieved by determiningtheir 16S rRNA sequence, and with strains from the related group ofsubtilis through their gyrA and cheA sequence. The isolation of thechromosome DNA of exponentially growing bacillus cells, DNAamplification and sequencing was conducted in accordance with Idriss etal. 2002. Here, the following primers were used to amplify the DNAsequences using polymerase chain reaction (PCR):

pRB1601: 5′GGATCCTAATACATGCAAGTCGAGCGG pRB1602:5′GGATCCACGTATTACCGCGGCTGCTGGC gyrFW: 5′CAGTCAGGAAATGCGTACGTC gyrRV:5′CAAGGTAATGCTCCAGGCATT cheAFW: 5′GAAACGGAKAYATGGMAGTBACMTCARACTGGCTGcheARV: 5′TGCTCRAGACGCCCGCGGWCAATGACAAGCTCTTC

An overview of the isolates obtained and their taxonomic classificationon the basis of their 16S rRNA sequence is shown in Table 2.

TABLE 2 DNA sequences of cold-adapted bacillus isolates from the Tibetanuplands and the Alpine foothills (altitude 1,400 m) Similarity withStrain Place of isolation 16SrRNA PGP¹ Growth ABI02S1 Grass roots, B.simplex + 04-45° C. Sejila Mountain, 338/358 (94%) Nyingtri, Tibet(pRB1601) B. simplex 403/464 (87%) (pRB1602) ABI02A1 Grass roots, B.atropheus + 10-45° C. Nyingtri, Tibet 450/458 (98%) (pRB1601) B.atrophaeus 572/590 (97%) (gyrFW) B. atrophaeus 733/757 (97%) (gyrREV)ABI02P1 Namtso Lake, B. pumilus + 10-50° C. Lhasa, Tibet 457/468 (98%)(pRB1602) ABI03 Grass roots, B. atropheus + 10-45° C. Alpine foothills,446/465 (96%) height 1,400 m (pRB1601) 474/475 (99%) B. atrophaeus608/625 (97%) (gyrFW) (pRB1602) ABI05 Grass roots, Bacillus atrophaeus +10-45° C. Alpine foothills, 450/458 (98%) height 1,400 m (pRB1601)447/478 (94%) (pRB1601) ABI12 Grass roots, B. simplex + 04-45° C. Alpinefoothills, 450/472 (95%) height 1,400 m (pRB1601) 461/472 (98%)(pRB1602) ¹PGP = plant growth promoting effect.

Example 2: Growth Characteristic of Cold-Tolerant Bacteria

The growth attempts were conducted in LB medium at differenttemperatures. Table 2 provides an overview of the growth behaviour ofthe cold-tolerant strains. The upper growth limit of B. simplex ABI02S1and ABI12, and B. atrophaeus ABI02A1 ABI03 and ABI05 was 45° C., whileB. pumilus ABI02P1 also grows at 50° C. B. atrophaeus and the mesophileB. amyloliquefaciens subsp. plantarum FZB42 were cultivated at 20° C.and 25°. At these temperatures, the cold-tolerant B. atrophaeus strainhad a faster growth rate than FZB42 (FIG. 1).

B. atrophaeus ABI02A1 was cultivated at different temperatures between20° and 50° C. (FIG. 1). The growth optimum was determined at 33-35° C.At 30° C., a slight deceleration of growth speed was registered. Below30° C., this effect was reinforced (25° C. and 20° C.). At 40° C.,growth was significantly delayed. At 50° C., a lysis of the cells wasalso observed (FIG. 2). The results confirm that in the case of B.atrophaeus ABI02A1, this is a mesophile, but also cold-tolerant strain,which despite a growth optimum of 33-35° C. also grows well at lowtemperatures.

The physiologipal properties of the cold-tolerant Bacillus atrophaeusand Bacillus simplex strains are described in examples 3 and 4 below.

Example 3: Physiological Properties of Bacillus atrophaeus

TABLE 3 Physiological properties of Bacillus atrophaeus ABI02A1, ABI03and ABI05 The use of sugars Glucose + Saccharose + Lactose − Maltose ±Fructose + Voges Proskauer + Exoenzyme formation Lipase (tributyrin) +Amylase (starch) + Protease (casein) + Keratinase (keratin) − Cellulase(cellulose) − Egg yoke test (lecithinase) − Pigment formation +Inhibition by antibiotics (flake test) CM5 chloramphenicol + KM5kanamycin +++ ERI erythromycin ++ Li25 lincomycin + AP100 ampicillin ++RIF 25** rifampicin +++ SPEC 100 spectinomycin ++ Bleo1 bleomycin +

In contrast to B. amyloliquefaciens subsp. plantarum FZB42, Bacillusatrophaeus ABI02A1, ABI03 and ABI05 also grow at 10° C. At 20° C. and25° C., ABI02A1 also has a higher growth speed than FZB42 (FIG. 2). Inthe agar diffusion test, ABI02A1, ABI03 and ABI05 inhibit Bacillussubtilis, Bacillus megaterium, and the phytopathogens Erwinia amylovora,Xanthomonas oryzae, Fusarium oxysporum, Rhizoctonia solani andSclerotinia scierotiorum.

Example 4: Physiological Properties of Bacillus simplex

B. simplex ABI02S1 and ABI12 have the following physiological properties(Table 4).

TABLE 4 Physiological properties of ABI02S1 and ABI12 Cell form Rod,0.9-1.0 × 3.0->4.0 m Endospores Ellipsoid Spore parent cell Not swollenUse of citric acid + Use of propionic acid − Catalase + Nitratereductase (NO₂ from NO₃) + Phenylalanin desaminase − Arginin dihydrolase− Indole formation − Anaerobic growth − Voges Proskauer (acetoneformation) − pH in Voges Proskauer medium 6.2 Lecithinase (egg yokereaction) − Growth at 50° C. − Growth at 45° C. (+) Growth at 40° C. +Growth at pH 5.7 − Growth at 2% NaCl + Growth at 5% NaCl − Growth at 7%NaCl − Growth at 10% NaCl − Acid formation of D-glucose + Acid formationof L-arabinose + Acid formation of D-xylose + Acid formation ofD-mannite + Acid formation of D-fructose + Gas of D-glucose − Hydrolysisof starch + Hydrolysis of gelatine + Hydrolysis of casein + Hydrolysisof Tween 80 + Hydrolysis of aesculin (+)

The physiological properties largely confirm the assignment to Bacillussimplex, but are not typical for Bacillus simplex for all features. Theanalysis of the cellular fatty acids shows a typical profile for thebacillus type.

B. simplex ABI02S1 and ABI12 grow in LB medium at 10° C., a temperatureat which B. amyloliquefaciens FZB42 can no longer grow. In furtherexperiments, it was shown that these two B. simplex strains can alsogrow at a temperature of 4° C. The growth maximum was 45° C.

In the agar diffusion test, B. simplex had a suppressive effect on thepathogenic funghi Rhizoctonia solani and Fusarium oxysporum, and onXanthomonas otyzae.

Example 5: Fermentation of Bacillus atrophaeus ABI02A1 and theProduction of a Spore Suspension

For example, a′ description is given here for the production of a sporesuspension for B. atrophaeus ABI02A1. The fermentation of B. atrophaeuswas conducted in a conventional stirring fermenter with a base volume of1.4 l medium under the following conditions:

Medium: Full medium with organic N and C source: Soya flour or maizesteeping liquor, low fat milk powder, yeast extract, salts

Sterilisation of the medium: 20 mins. at 121° C.

Template anti-foam agent: 200 ml

Stirrer speed: 700 RPM.

Fermentation temperature: 33° C.

Ventilation: 0.7 l/min (40% O₂ saturation)

pH: with NaOH set to 6.9

After 16 h, a maximum cell density of 1×10¹⁰ cells/ml was achieved. Theculture was continued in order to achieve the most complete possiblesporing of the vegetative cells. After 40 h, a spore titer of 3×10⁹ wasachieved. The spores were centrifuged out and transferred to 10% of theoriginal medium volume, under addition of propandiol (finalconcentration: 5%).

Example 6: Promotion of Germination of Maize Seeds Through Cold-TolerantBacillae

10 ml of a bacillus spore suspension were mixed with 30 ml 1%carboxymethyl celluluse (CMC), which served as an adhesive for improvedadhesion of the spores to the maize surface. The final concentration ofthe bacillus spores in the suspension was 1×107, 1×106, 1×105, 1×104cfu/ml. The maize seeds were surface-disinfected with 75% ethanol, 5%NaClO for 5 mins., before they were treated with the different dilutionsof the bacillus spore suspension for 5 mins. In each case, 3×10 seeds ofa spore concentration were laid out in a petri dish with damp filterpaper. The germination rate was determined after a week of darkincubation at 30° C. As a control, maize grains were used which had beentreated with a sterile nutrient medium+CMC.

A clear increase in germination speed was determined with theapplication of B. simplex and B. atrophaeus (FIG. 4).

Example 7: Growth Experiments with Arabidopsis thaliana

The roots of a 6-day-old Arabidopsis thaliana seedling were incubatedfor 5 mins. in a spore suspension (10⁵ spores/mil) of cold-adaptedbacillae. For comparison, a treatment with a spore suspension of FZB42,which is known for its growth-promoting effect, was conducted. Then, thetreated seedlings were transferred to Murashige-Skoog (MS) agar (1%).The quadratic plates (12×12 cm) were closed with parafilm and kept forthree weeks at 23° C. (8/16 light-dark rhythm). Then, the fresh weightof the Arabidopsis plants was determined. Cold-tolerant members of thetypes Bacillus simplex, Bacillus atrophaeus and Bacillus pumilus showeda clear phytostimulatory effect (FIG. 5, FIG. 6).

Example 8: Potted Test with Potato Plants in a Greenhouse

The test was conducted at the test facility for potato research, AgroNord, D-18190 Groβ-Lüsewitz as a potted test from 11.07.-11.10. 2011.The tubers of the potato test plant, of type “Burana”, was infested withsymptoms of a Rhizoctonia solani attack (“black scurf”, “pocks”). Thetubers were “stained” before planting with a diluted spore suspension ofthe bacillus formulae. The planting was conducted after the tubers haddried. The potatoes were harvested three months after planting. Therating of the potato plants and tubers showed a reduction of theRhizoctonia infestation compared to the control of 40-45% with thevariants treated with Bacillus pumilus ABI02P1 and Bacillus simplexABI02S1. At the same time, a yield increase of 95% (ABI02P1) and 57%(ABI02S1) was achieved (FIG. 7). The results achieved with the use ofthe cold-tolerant strains are thus comparable with the results achievedwith the use of FZB42. Here, it should be taken into account that thetest was conducted in the summer and early autumn, when the averagedaytime temperatures are by no mewls optimal for the use ofcold-tolerant bacteria.

Example 9: Field Test, Potatoes with B. pumilus and B. simplex

See the example below for information on the test procedure. Thestaining of the potato tubers with the mesophile, plant growth promotingbacterium FZB42 led to an increased yield compared to the untreatedcontrol of 14% (quantity used: 1 l spore suspension/ha). The increase inyield through staining with the cold-tolerant Bacillus pumilus ABI02P1(quantity used: 1 l spore suspension/ha) was 6% compared to theuntreated control. No increase in yield was achieved with the use of aspore suspension of the cold-tolerant Bacillus simplex ABI02S1 (FIG. 8).When interpreting the results, it should be taken into account that thetest was conducted from May to September, when the average daytimetemperatures are by no means optimal for the use of cold-tolerantbacteria.

Example 10: Field Test: The Use of Bacillus atrophaeus (ABI02A1) Leadsto an Increase in Yield with Potatoes

The field test was conducted by Agro Nord, Prüstelle fürKartoffelforschung, 18190 Groβ Lüsewitz, in Sanitz (Mecklenburg-WestPomerania). Preceding crop: maize. Soil type/number IS/35,fertilisation: 140 kg N. The potato type “Verdi” (medium-late-late) wasused. The spore suspension was applied using injection (lot injectionPL1, nozzle type: Flat jet nozzles TJ 800 15). Here, the sporesuspensions used (2×10¹³ spores/I) were diluted in 300 l of water beforeuse. The “staining” was conducted on 05.05. 2013 in the storagebuilding. Following successful drying of the tubers, they were plantedon 06.05.2013 at a soil temperature of 15.8° C.

Climatic conditions: The month of May was too warm by 1.2° C., and toodamp by 42.5 mm (79%). These were good emergence conditions for thepotatoes. The month of June generally matched the average valuesrecorded for a period of many years. However, precipitation primarilyoccurred in the form of heavy rain (13th=26 mm; 20th=14.3 mm; 25th=13.5mm) approx. 74% of the precipitation total for the month. The onlysignificant precipitation in July occurred on 3rd July with 28.4 mm; itwas followed by a very long dry period, which continued until the end ofAugust combined with too high temperatures.

The test was influenced by the occurrence of the phytopathogen, groundlevel fungus Rhizoctonia solani. Rhizoctonia solani J. G. Kühn is theamorphous form of Thanatephorus cucumeris (A. B. Frank) Donk(teleomorph), a basidiomycete from the Agaricales family. It leads toyield losses (loss of emergence, necroses on stalks and stolons, manysmall or misshapen tubers) and quality losses (potato pocks, “dry core”symptoms). The antagonistic effect of B. atrophaeus, FZB42 and thechemical fungicide Monceren Pro was determined (FIG. 10).

The use of a spore suspension (2×10¹⁰ cfu/ml) of Bacillus atrophaeus(ABI02A1) in a concentration of 1.0 l/ha leads to a reduction in diseasesymptoms of “black scurf” (dry core) and an increase in yield of 10%.Thus the effect of this organic phytostimulator corresponds to that ofthe chemical fungicide Monceren Pro (concentration: 1.5 l/ha).

Example 11: Field Test: Confirmation: The Use of Bacillus atrophaeus(ABI02A1) Leads to an Increased Yield for Potatoes and a ReducedInfestation of Rhizoctonia solani (Kuerzinger 2014)

In a second test series (Kürzinger 2014), the results of example 10 wereconfirmed. The effect of B. atrophaeus ABI02A1 spore suspensions wascomparable with that of the chemical fungicide Monceren (FIG. 12 andFIG. 13).

LEGEND FOR THE FIGURES

FIG. 1: Comparison of the growth of B. atrophaeus ABI02A1 and themesophile strain FZB42 at 20° C. Viand 25° C.

FIG. 2: Growth of B. atrophaeus ABI02A1 at different temperatures. Thegrowth optimum was determined at 33-35° C.

FIG. 3: Phase contrast: Bacillus simplex ABI02S1. The distal, ovalendospores are clearly visible in the non-swollen spore parent cells.

FIG. 4: Improved germination of Zea mays following incubation with B.simplex ABI02A1 (bottom row) compared to the control.

FIG. 5: Growth stimulation (in g fresh weight) of rabidopsis thalianathrough spore suspensions of cold-tolerant Bacillus strains. Incomparison: The effect of B. amyloliquefaciens FZB42. Control: withoutinoculation of Bacillus spores. The columns show the average value ofthree independent tests.

FIG. 6: Stimulation of root growth of Arabidopsis thaliana by B.atrophaeus ABI02A1 (upper left), and B. simplex ABI02S1 (upper right).CK=Control without inoculation with bacteria

FIG. 7: Potted test with potato plants (Kurzinger GmbH 2011). Comparisonof the tuber yield with untreated control. Application of 1 l sporesuspension per ha of FZB42 and B. pumilus ABI02P1. The spore suspensionof B. simplex ABI02S1 showed a lower concentration by a factor of 5, andwas applied in a correspondingly higher quantity (5 l/ha) to the potatoplants.

FIG. 8: Application of B. pumilus ABI02P1 (1 L/ha) and B. simplexABI02S1 to potato plants. Single application through staining with thespore suspension (FZB42 and Bacillus pumilus ABI02P1: 2×10¹⁰ cfu/ml,Bacillus simplex ABI02S1: 4×10⁹ cfu/ml) before planting)

FIG. 9: Randomised location map: 1 (control, no addition), 2 chemicalfungicide (Monceren Pro) 1.5 l/ha), 3 FZB42 (0.5 l/ha), 4 FZB42 (1.0l/ha), 5 FZB42 (2×0.5 I/ha), 8 ABI02A (1 l/ha). For each variant, fourrepetitions were conducted. The lot size was as follows: 6.90 m×3.00m=20.70 m², space between rows: 75 cm, space between tubers in the row:30 cm. “Staining” of the tubers in the storage building: 0.5.2013, dateplanted: 06.05.2013, emergence date: 02.06. 2013, harvest: 30.08.2013,treatment: 07. 10. 2013.

FIG. 10: Reduction in disease symptoms (“black scurf”) followingapplication of chemical fungicides (Monoceren Pro) and organicstimulators. The use of ABI02A ‘Bacillus atrophaeus’ (1.0 l/ha) leads toa considerable reduction in disease symptoms.

FIG. 11: The use of Monoceren (chemical fungicide) and spore suspensionsof FZB42 and ABI02A1 (Bacillus atrophaeus) among potatoes of type Verdi(Sanitz, Kürzinger 2013) leads to an increase in yield (%). Quantitiesused: 1.0=1.0 kg/ha.

FIG. 12: The application of Bacillus atrophaeus ABI02A1 leads to aconsiderable increase in yield, which can be compared to that of thechemical fungicide Monoceren. With the mesophile strain FZB42, asomewhat higher yield increase was achieved. Quantities of fungicidesused: 1.5 l/ha (Monceren), 0.5 1.0 l/ha and 2×0.5 l/ha with sporesuspensions of FZB42 and ABI02A1.

FIG. 13: At the same time, the infestation of sclerotia (Rhizoctoniasolani) was reduced with the application of Bacillus atrophaeus ABI02A1compared to the untreated control. Illustration of all infestationclasses (% sclerotia infestation) with the use of Monoceren, FZB42 andB. atrophaeus. B. atrophaeus leads to a reduction in sclerotiainfestation compared to the untreated control. Infestation classes: 0,<1, 1.1-5, 5.1-10, 10.1-15 and >15.

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1. A method of stimulating the growth of cultivated plants, comprisingtreating said plants with a cold-tolerant Bacillus strain.
 2. The methodaccording to claim 1, wherein the cold-tolerant Bacillus straincomprises a cold-tolerant strain of the Bacillus atrophaeus or Bacillussimplex species.
 3. The method according to claim 1, wherein thecold-tolerant Bacillus strain comprises a cold-tolerant strain Bacillusatrophaeus ABI02A1 DSM 32019, ABI03 DSM 32285, or ABI05DSM 29418, or amixture thereof.
 4. The method according to claim 1, wherein thecold-tolerant Bacillus strain comprises a cold-tolerant strain Bacillussimplex ABI02S1 DSM 32020 or ABI12 DSM 32283, or a mixture thereof. 5.The method according to claim 1, wherein the cold-tolerant Bacillusstrain comprises a cold-tolerant Bacillus strain comprising Bacillusatrophaeus ABI02A1 DSM 32019, ABI03 DSM 32285, or ABI05 DSM 29418, or amixture thereof; and a cold-tolerant Bacillus strain comprising Bacillussimplex ABI02S1 DSM 32020 or ABI12 DSM 32283, or a mixture thereof. 6.The method according to claim 1, wherein the cold-tolerant Bacillusstrain is formulated as a fluid spore suspension.
 7. The methodaccording to claim 1, wherein said treating comprises treating seedsduring planting or treating seeds after planting.
 8. The methodaccording to claim 1, wherein the cold-tolerant Bacillus strain isformulated as a dry preparation (“dry stain”).
 9. The method accordingto claim 1, wherein the cold-tolerant Bacillus strain further compriseshumic acid.
 10. The method according to claim 1, further comprising amesophile plant growth promoting bacteria of the Bacillales group. 11.The method according to claim 1, further comprising a plant growthpromoting fungus Trichoderma sp.
 12. The method according to claim 6,wherein the fluid spore suspension comprises at least 2×10⁹ spores/ml.13. The method according to claim 6, wherein the fluid spore suspensioncomprises at least 1×10¹⁰ spores/ml.
 14. The method according to claim7, wherein said treating is by pour administration or by sprayadministration.
 15. The method according to claim 8, wherein theformulation comprises at least 5×10⁹ spores/ml.
 16. The method accordingto claim 8, wherein the formulation comprises at least 2×10¹⁰ spores/ml.17. The method according to claim 10, wherein said mesophile plantgrowth promoting bacteria of the Bacillales group comprises a Bacillusbacteria or a Paenibacillus bacteria.
 18. The method according to claim17, wherein said Bacillus bacteria comprises a Bacillusamyloliquefaciens ssp. plantarum.